Transcription factor activation and mitogenic synergism in airway smooth muscle cells.

Simultaneous treatment of human airway smooth muscle (HASM) cells with lysophosphatidic acid (LPA) and epidermal growth factor (EGF) leads to strikingly synergistic stimulation of mitogenesis. The purpose of this study was to explore potential sites for signal integration mediating synergism, focusing on extracellular signal-regulated kinase (ERK) and transcription factors involved in proliferation and inflammation as likely candidates. Activation of ERK was analysed by immunoblotting. Transcription factor activation was assessed using HASM cells transduced with luciferase reporter gene constructs. LPA and EGF both activated ERK but had no synergistic effect when combined. LPA and EGF both activated activator protein (AP)-1, cyclic adenosine monophosphate response element-binding protein, nuclear factor of activated T-cells and the serum response element; however, only AP-1 activation exhibited synergism. Activation of the inhibitory guanine nucleotide-binding protein and of ERK signalling pathways were required for most transcription factor responses to LPA. In contrast, nuclear factor (NF)-kappaB was activated by LPA but not EGF and NF-kappaB activation was completely blocked only when Rho was inhibited. Rapid activation of Rho was observed in response to LPA but not to EGF. Importantly, inhibition of Rho selectively blocked synergism in both AP-1 activation and mitogenesis. In summary, extracellular signal-regulated kinase activation is required for many transcription factor responses to lysophosphatidic acid and epidermal growth factor, however it is not synergistic. Activation of activator protein-1 is synergistic, and Rho activation by lysophosphatidic acid is required for synergism in both activator protein-1 activation and mitogenesis.

Dual effects of lysophosphatidic acid on human airway smooth muscle cell proliferation and survival.

Lysophosphatidic acid (LPA) is a phospholipid growth mediator found in serum at 2-20 microM. In many cell types, including human airway smooth muscle (HASM) cells, LPA-induced proliferation occurs at 10-100 microM LPA. At these concentrations LPA forms Ca2+ precipitates. The potential involvement of Ca2+ and Ca2+ LPA precipitates in LPA-induced HASM cell mitogenesis was investigated. In the absence of extracellular Ca2+, 10 and 30 microM LPA stimulated HASM cell mitogenesis. However, with 100 microM LPA in the absence of extracellular Ca2+, HASM cells exhibited a profound shape change and loss of viability, determined to be apoptosis by both DNA staining and assessment of cytosolic nucleosomal reactivity. A bioassay based on the adenosine 3':5'-cyclic monophosphate response of C62B rat glioma cells was used to measure the bioactivity of LPA solutions prepared in Ca2+ free and Ca2+ containing medium. After 24 h, a 100 microM LPA solution in Ca2+ free medium contained markedly greater bioactivity than a 100 microM LPA solution made in Ca2+ containing medium. In summary, formation of Ca2+ LPA precipitates decreases the amount of biologically active LPA in solution, and high concentrations of bioactive LPA achieved in Ca2+ free but not in Ca2+ containing medium induce apoptosis of HASM cells.

Synergistic stimulation of airway smooth muscle cell mitogenesis.

Previous studies showed that human airway smooth muscle (HASM) cells treated with lysophosphatidic acid (LPA), a pertussis toxin (PTX)-sensitive G protein-coupled (GPC) mitogen, simultaneously with epidermal growth factor (EGF), a receptor tyrosine kinase (RTK) mitogen, exhibit markedly synergistic stimulation of mitogenesis. We now show that the RTK mitogens basic fibroblast growth factor, insulin-like growth factor-1, insulin, platelet-derived growth factor-AA, and platelet-derived growth factor-BB, as well as transforming growth factor-beta, all induced synergistic stimulation of mitogenesis in the presence of LPA. The PTX-sensitive GPC mitogens carbachol and endothelin-1 and the PTX-insensitive GPC mitogens sphingosine-1-phosphate and thrombin exhibited synergistic stimulation together with EGF. Several RTK-RTK growth factor pairs and GPC-GPC mitogen pairs were also synergistic. HASM cells showed synergistic responses to serum plus EGF but not to serum plus LPA. Testing various other cell types showed that synergism between LPA and EGF occurred in other smooth muscle cells because both vascular smooth muscle cells and mesangial cells exhibited synergism. Additionally, human fetal lung fibroblasts also showed striking synergism. These results indicate that HASM cells can respond synergistically to a wide variety of mitogen combinations and that this synergism is a feature shared with other contractile cell types.